Viral infectivity in paediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, although they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in paediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virological features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children with coronavirus disease 2019 (COVID-19), we compared viral RNA levels (clinical RT-qPCR C T) and infectious virus titres from 144 SARS-CoV-2-positive clinical samples collected from children aged 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.


INTRODUCTION
During the early months of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, notable uncertainty emerged regarding the role of children in transmission dynamics [1]. With time, it became clearer that children were susceptible to infection with SARS-CoV-2, but that the vast majority of children experienced mild symptoms with lower incidence of severe disease [2]. This pattern remained consistent despite the later emergence of SARS-CoV-2 variants, including delta and omicron, even among children <5 who were ineligible for vaccination [3]. The relative lack of severe disease in the paediatric population raised questions regarding viral kinetics and infectivity in children versus adults.
We hypothesized that unique virological features (i.e. defective interfering genomes or other changes in particle infectivity) in children could explain this apparent decrease in symptoms and transmissibility early in the pandemic. The infectious virus to viral RNA ratio varies greatly in RNA viruses, including SARS-CoV-2, which has a ratio of 10 3 : 1 to 10 6 : 1 [4]. This high ratio means that most genome copies are associated with non-infectious particles, thus leaving room for potential changes in the ratio between infectious virus and viral RNA across different age groups. Changes in this ratio have been observed between SARS-CoV-2 variants; both the epsilon and delta variants had significantly higher infectious virus to viral RNA ratios than the alpha variant [5]. Children have been found to produce high levels of viral RNA, but early in the pandemic infectious virus titre was not commonly measured. Plaque or focus assays that directly measure the number of infectious OPEN ACCESS virions are the gold standard for determining viral load. However, they require inoculating a susceptible cell line with serial dilutions of the sample of interest, allowing virus to infect cells under a semi-solid overlay, and detection of individual 'foci' of infection by the viral-specific antibodies or the presence of cytopathic effect. Due to the challenges posed by measurement of infectious viral titres (including the requirement for BSL-3 containment), the majority of work examining viral loads in clinical samples has measured viral RNA levels, as determined by RT-qPCR cycle threshold (C T ). A previous study using this technique reported no differences in viral RNA load in adults and children, when controlling for the presence of symptoms [6]. A second study reported both RNA viral load and level of infectious virus using a semi-quantitative method (TCID 50 ) in paediatric clinical samples, which revealed that neither age nor disease severity impacted on viral load [7]. In contrast, however, others have found that children have decreased levels of viral RNA, lower infectious virus titres as measured by TCID 50 and are less likely to have virus successfully isolate in cell culture as compared to adults [8], although paediatric delta variant infections increase the culture-positive viral titres to comparable levels to adults [9]. Other work indicates that ancestral SARS-CoV-2 replicates less efficiently in both children and paediatric versus adult nasal epithelial cells, a defect that omicron was able to abolish [10]. Finally, we and others have demonstrated a dynamic relationship between C T values and infectious viral titres with potential for significant discrepancies and a ratio dependent on both viral and host factors [5], but this work did not include children [11].
Therefore, to further understand SARS-CoV-2 infection in children, we investigated the ratio of infectious virus titre to RNA viral load in children aged 0 to <18 years old. We hypothesized that the ratio of infectious virus to RNA viral load would be positively associated with age.

Sample selection
Banked SARS-CoV-2-positive nasopharyngeal specimens from children aged 0 to <18 years old collected and stored at Children's Wisconsin, Milwaukee, Wisconsin, USA between 14 September 2020 and 17 May 2021 were identified. Deidentified samples were binned into four age groups (<1, 1-5, 6-11 and 12-17) and stratified by clinical C T value (<20, 20-24, 25-29 and 30-34) to select a sample of children representing the full spectrum of both ages and C T values (due to the deidentified nature of the samples, information on biological sex and other metadata was not available).

RNA extractions and RT-PCR
Total nucleic acid was extracted on the NucliSENS easyMAG or EMAG automated extraction instruments (bioMérieux). SARS-CoV-2 RNA was detected using previously published primers/probes for the SARS-CoV-2 E gene (Sarbeco [12]) on the 7500 Fast Real-Time PCR System or QuantStudio 7 Pro platforms.

Viral titre
All SARS-CoV-2 viral titring was conducted at the University of Vermont BSL-3 facility under an approved IBC protocol. Clinical samples were titred using a microfocus forming assay on VeroE6-TMPRSS2 cells (Japanese Cancer Research Resources Bank #JCRB1819). Cells were seeded 24 h before infection, 60 000 cells/well. Samples were serially diluted using 10-fold dilutions. All samples were titred in technical duplicate, except for the neat dilution due to insufficient sample volume. Cells were infected for 1 h at 37 °C, and then overlaid with 1.2 % methylcellulose in Dulbecco's Modified Eagle Medium (DMEM) (Gibco cat. 11965084) and incubated for 24 h at 37 °C, 5 % CO 2 . Cells were fixed using 4 % formaldehyde in phosphate-buffered saline (PBS). After fixation, cells were permeabilized using 0.01 % Triton X-100 in PBS for 15 min and then incubated in a primary, cross-reactive rabbit anti-SARS-CoV N monoclonal antibody (Sino Biological 40143R001100) at 1 : 20 000 dilution. Cells were washed in PBS and then incubated in a peroxidase-labelled goat-anti-rabbit antibody (Seracare 5220-0337) at 1 : 4000, followed by the peroxidase substrate (SeraCare, cat. #5510-0030). Foci were imaged using a BioTek Cytation instrument and counted manually. Samples were titred in a blinded fashion; personnel performing BSL-3 titring did not have access to sample C T s.

Statistical analysis
Viral titres were log-transformed for analysis. Linear regression was used to predict log titre as a function of C T , fitting separate models without age and to control for continuous and categorical age effects, and no data points were excluded from analysis. Models were compared by F test. Data were analysed and plotted with R.

RESULTS
n=144 clinical specimens were selected to determine the relationship between the infectivity of SARS-CoV-2 in paediatric samples and RNA viral load. As expected, higher RNA viral load generally correlated with higher infectious virus titre, Fig. 1. SARS-CoV-2 viral infectivity does not vary by age in a paediatric population. (a) A set of 144 clinical samples from children infected with SARS-CoV-2 was used to examine the relationship between infectious virus titre and RNA viral load as a function of patient age. Individual specimen measurements of E gene RNA levels (C T ) on the x-axis are plotted against viral titre, as measured in focus forming units (f.f.u. ml −1 ) on the y-axis. Dashed line indicates the limit of detection for infectious titre (20 f.f.u. ml −1 ). Samples for which we could not measure a viral titre were assigned fixed values of one-tenth the limit of detection (2 f.f.u. ml −1 ). Lines of best fit were generated by linear regression on log-transformed titre data as a function of C T and age group. Symbol and colour indicate age group (<1, purple circle; 1-5, blue square; 6-11, turquoise diamond; 12-17, green triangle). although as reported previously this ratio was somewhat variable (Fig. 1a) [5,11]. In linear regression, the relationship between infectious viral titre and C T was not significantly modified by age (P=0.156) or age group (P=0.355 overall by F test). These data indicate that there is no difference in the infectiousness of SARS-CoV-2 produced by children, regardless of age.

DISCUSSION
Consistent with previous findings, we found no significant differences in the relationship between SARS-CoV-2 infectious virus titre and RNA viral load in children across the paediatric age spectrum [6, 7]. Our findings suggest equal levels of viral infectivity in young children and adolescents with similar RNA viral loads, thus other factors are contributing to differential disease severity in this population. Interestingly, while we and others have found equal levels of viral infectivity across paediatric age groups, it has been found that age does influence severity of disease within the paediatric population. In one cohort, asymptomatic patients and those with mild disease were significantly younger than those hospitalized, suggesting that age-related factors other than viral infectivity are playing a role in disease manifestation [7]. This could include differences in immune response (i.e. heavier reliance on the innate immune response), fewer comorbidities, or even fewer viral entry receptors than adults [13]. The limitations of this study include lack of access to viral sequencing and individual-level metadata, which could reveal differences in infectivity as a result of viral genetic background, days post-symptom onset, host immune status and vaccination status. Furthermore, as sample collection took place at a children's hospital there was no direct comparison with adult samples, although we did include samples in older teens who would closely resemble adults biologically. While our results are in agreement with several recent studies, replicating prior work in a novel cohort with independent methodologies is an important step in establishing a clear understanding of SARS-CoV-2 infection dynamics in children. While we only had access to samples early in the pandemic (during the circulation of alpha and prior to the appearance of variants of concern; Fig. 1b), future studies investigating the viral infectivity in paediatric patients during waves of delta, omicron and future variants would strengthen our understanding of the dynamics of SARS-CoV-2 infection in the paediatric population. Regardless, the finding that children of all ages with confirmed SARS-CoV-2 infection are capable of producing high levels of replication-competent virus should be balanced against the finding that there was no age-dependent effect, when designing public health policy-based interventions.

Funding information
The authors received no specific grant from any funding agency.   Comments: Thank you for submitting your revised manuscript for publication in Access Microbiology. After reviewing the manuscript, I'm happy that all reviewer's comments have been addressed. However, some minor changes are needed to include: 1. the PFU abbreviation in full when first mentioned (line 53) 2. use either the genome to PFU ratio or higher infectious virus to RNA ratio rather than using these interchangeably (lines 53-59) 3. correct some minor spelling and grammar mistakes e.g. asymptotic on line 150

Author response to reviewers to Version 1
We would like to thank the Editor and the Reviewers' for their time and thoughtful suggestions to improve this manuscript. We have included an additional figure panel (Fig 1B) as well as additional references and further discussion to address these comments.

Presentation of results
The methods and results sections are clear and concise.
3. How the style and organization of the paper communicates and represents key findings 4. Literature analysis or discussion 1. The rationale for investigating whether infectivity is associated with lower incidence of severe disease in pediatric population should be made clear in the introduction.
To this end, the author should include (if possible) more references that support this hypothesis. Currently, only one of the papers referenced in the introduction supports an association between viral RNA load and age (ref.7).
We agree that the rationale for investigating viral infectivity should be expanded upon. We have added additional justification for our investigation starting at line 53, "The genome to PFU ratio varies greatly in RNA viruses, including SARS-CoV-2, which has a ratio of 10 3 :1 to 10 6 :1 4

. This high genome to PFU ratio means that most genome copies are associated with non-infectious particles, thus leaving room for potential changes in the ratio between infectious virus and viral RNA across different age groups. Changes in this ratio have been observed between SARS-CoV-2 variants; both the Epsilon and Delta variants had significantly higher infectious virus to RNA ratios than the Alpha variant 5 . Children have been found to produce high levels of viral RNA, however, early in the pandemic, infectious virus titer was not commonly measured. "
2. The results of ref 5 (line 79) should be discussed, or at least mentioned.
We have included discussion of reference 5 in line 69, "A second study reported both RNA viral load and level of infectious virus using a semi-quantitative method (TCID 50 ) in pediatric clinical samples, which revealed that neither age or disease severity impacted viral load" We have also included a discussion of the reference in line 155, "Interestingly, while we and others have found equal levels of viral infectivity across pediatric age groups, it has been found that age does influence severity of disease within the pediatric population. In one cohort, asymptotic patients and those with mild disease were significantly younger than those hospitalized, suggesting that age related factors other than viral infectivity are playing a role in disease manifestation 7 " 3. The sentence beginning line 79 should be associated with reference 7 only.
We agree and have removed references 5 and 6. 4. A discussion of other factors that could contribute to differential disease severity between children and adults (e.g. innate immunity) would make for a more well-rounded introduction.
We have added discussion of other potential factors that contribute to differential disease severity in line 159, "This could include differences in immune response (i.e. heavier reliance on the innate immune response), fewer comorbidities, or even fewer viral entry receptors than adults 13 " 5. In line 130, the statement "our findings suggest equal levels of viral infectivity in children and in adults with similar RNA viral loads" is inappropriate because there was no direct comparison with adult samples in this article.
We apologize for any confusion, the statement, "our findings suggest equal levels of viral infectivity in children and in adults with similar RNA viral loads" exists in a previous version of this paper that had been deposited to medRxiv, however the version submitted to Access Microbiology has removed the comparison to adults as we agree that the statement is inappropriate. See line 153, "Our findings suggest equal levels of viral infectivity in young children and adolescents with similar RNA viral loads." Any other relevant comments 6. The problem or question, as it is framed in the introduction, is essentially, "Why is there a lower prevalence of severe disease following SARS-COV-2 infection in children compared to adults?". The authors hypothesize that infectivity of the virus may be associated with this phenomenon. However they do not include a direct comparison to adult samples. By looking exclusively at samples from children (aged 0 to <18 years old), they can only conclude that infectivity does not change within childhood, which does not shed much light on the original problem / question.If the authors could provide some evidence or a reference supporting the idea that younger children have lower incidence of severe disease following sarscov2 infection compared to "older teens" that would strengthen the conclusion.
Line 155 now discusses a finding in which pediatric patients who were asymptomatic or experiencing mild disease were significantly younger than those hospitalized. "Interestingly, while we and others have found equal levels of viral infectivity across pediatric age groups, it has been found that age does influence severity of disease within the pediatric population. In one cohort, asymptotic patients and those with mild disease were significantly younger than those hospitalized, suggesting that age related factors other than viral infectivity are playing a role in disease manifestation 7 . This could include differences in immune response (i.e. heavier reliance on the innate immune response), fewer comorbidities, or even fewer viral entry receptors than adults 13 " Please rate the manuscript for methodological rigour Reviewer 2 Comments to Author: Thank you for the opportunity to review your work. In summary, the researchers sought to evaluate if there was a virologic correlate of clinical severity of disease and infectivity in children (0-18 years) for SARS-CoV-2/ COVID-19 by interrogating samples utilizing cell culture and comparing "viral load"/Ct values (from RT-PCR) to cell culture yield. Samples were obtained from Children's in Wisconsin from September 14, 2020 to May 17, 2021 (n = 144). Stratification by age groups and Ct values was performed and blinding by staff performing cell culture was also undertaken to reduce bias. Their finding was that though a relationship between viral titer and Ct was observed, there was no significant relationship between age and viral yield. Limitations are nicely outlined. The conclusion from their data that children, regardless of age, produce high levels of viable virus is justified.
We are pleased the reviewer agrees our conclusions are justified by our data, and hope this revised manuscript will be found suitable for publication.
I have a few suggestions to improve the work: 1. Overall the methods are appropriately described in terms of RT-PCR and cell culture. Based on the timeline of sample acquisition, I would assume that these isolates contained more ancestral SARS-CoV-2 and the Alpha VOC. Can the authors provide the epidemiological datato support those assumptions? Notably, the timeframe does not include Delta nor Omicron which are likely of more relevance in 2023. If available, including this data would add robustness and relevance.
We have created a figure showing the epidemiological data from samples sequenced in Milwaukee, Wisconsin during the time of sample acquisition for this study (see new Fig1B, and below). The reviewer is correct that these isolates were primarily ancestral SARS-CoV-2 and the Alpha variant. While we agree that further study of more recent variants would be interesting, we do not have access to these samples and feel that on-going study of later VOCs represents a body of work that is outside the scope of this paper. Finally, the first author on this manuscript is preparing to leave our lab to attend a PhD program and we do not have personnel or funds available to carry out additional work on this project.
2. It is possible to stratify by time from sampling to placement on cell culture? Was the yield significantly different between relatively fresh samples and older ones? Our work demonstrated reduced yield the longer it was from clinical sampling to cell culture.
We understand the concern and appreciate the reviewer sharing their results with us. We do not think this is likely to be a major confounder for our study, as our previously published data demonstrates that in the case of SARS-CoV-2, viral RNA and infectious virus remains stable through multiple freeze-thaw cycles, as well as when stored at 4°C and 24°C for up to two weeks (within normal limits for clinical sample processing). See PMID:34862752. Unfortunately we do not have access to specific metadata on time from sample collection to freezing for these samples, although all were stored at -80 for long term storage after collection, shipped on dry ice, and arrived frozen to our lab at UVM.
3. Though references were provided to support that there was no difference between age groups, SARS-CoV-2 viral loads/Ct and viable virus, there are also references that found a difference. Bullard et al. (CMAJ April 2021) demonstrated an age dependent difference. The same group also showed that though Ct values between adults and children were different, viable virus yield was not different for the Delta VOC (Garnett et al. Microbiol Spectr August 2022).
We thank the reviewer for this helpful suggestion, and we have added information from the additional studies suggested in our introduction section to highlight alternate findings regarding pediatric viral infectivity. See line 71 "In contrast however, others have found that children have decreased levels of viral RNA, lower infectious virus titers as measured by TCID 50 , and were less likely to have virus successfully isolate in cell culture as compared to adults 8 , however pediatric Delta variant infections increases the culture positive viral titers to comparable levels to adults 9 . " 4. The presentation of results is clear. I appreciate Figure 1 and it is formatted well.
We thank the reviewer for this observation.
5. The paper is organized and concisely and clearly conveys the key findings and background around the topic. The limitations are listed and are appropriate. I would add some of the limitations I mention above to the list if they cannot be rectified. As mentioned above, the authors could include additional studies that did find a difference. Overall, I appreciate the work and think the results do add to the historical understanding of viral dynamics of SARS-CoV-2 in children. With the addition of Delta and Omicron variants, the work would become much more relevant.
We thank the reviewer for their helpful comments and agree that the addition of data regarding the Delta and Omicron variants would be interesting and important for follow-up studies, however we are not able to include this data at this time (see point 1 above), so we have added this as a limitation in our discussion section. See line 169, "While we only had access to samples early in the pandemic (during the circulation of Alpha and prior to the appearance of variants of concern; Fig 1B) Comments: Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers' comments.

Anonymous.
Date report received: 19 January 2023 Recommendation: Major Revision Comments: Thank you for the opportunity to review your work. In summary, the researchers sought to evaluate if there was a virologic correlate of clinical severity of disease and infectivity in children (0-18 years) for SARS-CoV-2/COVID-19 by interrogating samples utilizing cell culture and comparing "viral load"/Ct values (from RT-PCR) to cell culture yield. Samples were obtained from Children's in Wisconsin from September 14, 2020 to May 17, 2021 (n = 144). Stratification by age groups and Ct values was performed and blinding by staff performing cell culture was also undertaken to reduce bias. Their finding was that though a relationship between viral titer and Ct was observed, there was no significant relationship between age and viral yield. Limitations are nicely outlined. The conclusion from their data that children, regardless of age, produce high levels of viable virus is justified. I have a few suggestions to improve the work: -Overall the methods are appropriately described in terms of RT-PCR and cell culture. Based on the timeline of sample acquisition, I would assume that these isolates contained more ancestral SARS-CoV-2 and the Alpha VOC. Can the authors provide the epidemiological data to support those assumptions? Notably, the timeframe does not include Delta nor Omicron which are likely of more relevance in 2023. If available, including this data would add robustness and relevance. -It is possible to stratify by time from sampling to placement on cell culture? Was the yield significantly different between relatively fresh samples and older ones? Our work demonstrated reduced yield the longer it was from clinical sampling to cell culture. -Though references were provided to support that there was no difference between age groups, SARS-CoV-2 viral loads/Ct and viable virus, there are also references that found a difference. Bullard et al. (CMAJ April 2021) demonstrated an age dependent difference. The same group also showed that though Ct values between adults and children were different, viable virus yield was not different for the Delta VOC (Garnett et al. Microbiol Spectr August 2022). -The presentation of results is clear. I appreciate Figure 1 and it is formatted well. -The paper is organized and concisely and clearly conveys the key findings and background around the topic. The limitations are listed and are appropriate. I would add some of the limitations I mention above to the list if they cannot be rectified. -As mentioned above, the authors could include additional studies that did find a difference. Overall, I appreciate the work and think the results do add to the historical understanding of viral dynamics of SARS-CoV-2 in children. With the addition of Delta and Omicron variants, the work would become much more relevant. Thank you.

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript

Very good
To what extent are the conclusions supported by the data? Partially support

Anonymous.
Date report received: 17 January 2023